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Deactivation of aminoglycosides by their modifying enzymes, including a number of aminoglycoside O-phosphotransferases, is the most ubiquitous resistance mechanism in aminoglycoside-resistant pathogens. Nonetheless, in a couple of biosynthetic pathways for gentamicins, fortimicins, and istamycins, phosphorylation of aminoglycosides seems to be a unique and initial step for the creation of a natural defensive structural feature such as a 3',4'- dideoxy scaffold. Our aim was to elucidate the biochemical details on the beginning of these C3',4'-dideoxygenation biosynthetic steps for aminoglycosides. The biosynthesis of istamycins must surely involve these 3',4'-didehydroxylation steps, but much less has been reported in terms of characterization of istamycin biosynthetic genes, especially about the phosphotransferase-encoding gene. In the disruption and complementation experiments pointing to a putative gene, istP, in the genome of wild-type Streptomyces tenjimariensis, the function of the istP gene was proved here to be a phosphotransferase. Next, an in-frame deletion of a known phosphotransferase-encoding gene forP from the genome of wild-type Micromonospora olivasterospora resulted in the appearance of a hitherto unidentified fortimicin shunt product, namely 3-O-methyl-FOR-KK1, whereas complementation of forP restored the natural fortimicin metabolite profiles. The bilateral complementation of an istP gene (or forP) in the ΔforP mutant ( or ΔistP mutant strain) successfully restored the biosynthesis of 3',4'- dideoxy fortimicins and istamycins , thus clearly indicating that they are interchangeable launchers of the biosynthesis of 3',4'-dideoxy types of 1,4-diaminocyclitol antibiotics.

The accumulation of rubber waste has become a major environmental issue worldwide due to its adverse effects on ecology. Bioremediation is focused to minimize this problem. In this study, the degradation of deproteinized natural rubber (DPNR) using bacterial consortia enriched from Sapa soil was examined. This soil was not history with rubber. The highest 39.16 ± 1.95 % weight loss of DPNR film was detected in the sixth enrichment consortium after 30 days of incubation. The occurrence of hydroxyl group in the film was observed by Fourier Transform Infrared Spectroscopy analysis. The changes in bacterial community in the consortia were determined by metagenomic analysis using 16S rRNA gene sequencing. The dominant phyla in all consortia were Actinobacteria, Bacteroidetes, and Proteobacteria, while the phylum Actinobacteria was key rubber-degraders in the consortia.

A high-performance liquid chromatography with electrospray ionization ion trap tandem mass spectrometry method was developed and validated for the robust profiling and characterization of biosynthetic congeners in the 2-deoxy-aminocyclitol istamycin pathway, from the fermentation broth of Streptomyces tenjimariensis ATCC 31603. Gradient elution on an Acquity CSH C18 column was performed with a gradient of 5 mM aqueous pentafluoropropionic acid and 50% acetonitrile. Sixteen natural istamycin congeners were profiled and quantified in descending order; istamycin A, istamycin B, istamycin A0, istamycin B0, istamycin B1, istamycin A1, istamycin C, istamycin A2, istamycin C1, istamycin C0, istamycin X0, istamycin A3, istamycin Y0, istamycin B3, and istamycin FU-10 plus istamycin AP. In addition, a total of five sets of 1- or 3-epimeric pairs were chromatographically separated using a macrocyclic glycopeptide-bonded chiral column. The lower limit of quantification of istamycin-A present in S. tenjimariensis fermentation was estimated to be 2.2 ng mL−1. The simultaneous identification of a wide range of 2-deoxy-aminocyclitol-type istamycin profiles from bacterial fermentation was determined for the first time by employing high-performance liquid chromatography with tandem mass spectrometry analysis and the separation of istamycin epimers. This article is protected by copyright. All rights reserved

To date, non-food vegetable oil has been considered as the primary source for biodiesel production. Rubber seed oil has high acid value (34 mgKOH/g) and can be used for biodiesel synthesis. The purpose of this study was to investigate esterification of fatty acid, which derived from rubber seed oil, in a plug flow reactor system at high temperature and low methanol consumption. Response surface methodology was applied for design experiment and optimization of esterification reaction. Temperature, methanol consumption, and sulfuric acid were chosen as variables to examine their influence in a conversion to methyl ester. At 140°C, at 5:1 methanol to fatty acid ratio (by mole), H2SO4 1.5 (%v/w), and space time 20 min, the conversion to methyl ester attained 98.2%. Fourier transform infrared spectroscopy (FTIR) and gas chromatography-Mass spectrometry (GC-MS) were used for analysis and to confirm the formation of methyl ester. Methyl ester was characterized for biodiesel fuel properties in accordance ...

In this study, granular sludge formation was carried out using an aluminum chloride supplement in an upflow anaerobic sludge blanket (UASB) reactor treating natural rubber processing wastewater. Results show that during the first 75 days after the start-up of the UASB reactor with an organic loading rate (OLR) of 2.65 kg-COD·m−3·day−1, it performed stably with a removal of 90% of the total chemical oxygen demand (COD) and sludge still remained in small dispersed flocs. However, after aluminum chloride was added at a concentration of 300 mg·L−1 and the OLR range was increased up to 5.32 kg-COD·m−3·day−1, the total COD removal efficiency rose to 96.5 ± 2.6%, with a methane recovery rate of 84.9 ± 13.4%, and the flocs began to form granules. Massively parallel 16S rRNA gene sequencing of the sludge retained in the UASB reactor showed that total sequence reads of Methanosaeta sp. and Methanosarcina sp., reported to be the key organisms for granulation, increased after 311 days of operation. This indicates that the microbial community structure of the retained sludge in the UASB reactor at the end of the experiment gave a good account of itself in not only COD removal, but also granule formation.

Natural rubber processing wastewater contains high concentrations of organic compounds, nitrogen, and other contaminants. In this study, a treatment system composed of a baffled reactor (BR), an upflow anaerobic sludge blanket (UASB) reactor, and a downflow hanging sponge (DHS) reactor was used to treat natural rubber processing wastewater in Vietnam. The BR showed good total suspended solids (TSS) removal of 47.6%, as well as acidification of wastewater. The UASB reactor achieved a high chemical oxygen demand (COD) removal efficiency of 92.7 ± 2.3% and energy recovery in the form of methane with an organic loading rate of 12.2 ± 6.6 kg-COD m−3 day−1. The DHS reactor showed high performance in residual organic matter removal from UASB effluent. In total, the system achieved high-level total COD removal of 98.6% ± 1.2% and TSS removal of 98.0% ± 1.4%. Massive parallel 16S rRNA gene sequencing of the retained sludge in the UASB reactor showed the predominant microbial phyla to be Bacteroidetes, Firmicutes, ...

Biodegradation is a potential way of decomposing deproteinized natural rubber (DPNR). The enrichment consortia were demonstrated from a rubber processing factor waste. Nine DPNR-degrading bacteria were isolated from those consortia. The highest DPNR film weight loss in a mineral salt medium (MSM) was 43.92 ± 2.30 % after 30 days incubation using strain 5A1. The formation of aldehyde group during rubber degradation of 5A1 was determined using Schiff staining and Fourier Transform Infrared spectroscopy (FTIR) analysis. The 16S rRNA gene sequence, of 5A1 showed the highest identity with that of Gordonia soli CC-AB07. This is the first report to demonstrate a strong ability to degrade DPNR by Gordonia sp.

We have developed a new computational scheme for high-accuracy prediction of the isotropic hyperfine coupling constant (HFCC) of heavy molecules, accounting for the high-level electron correlation effects, as well as the scalar-relativistic effects. For electron correlation, we employed the ab initio density matrix renormalization group (DMRG) method in conjunction with a complete active space model. The orbital-optimization procedure was employed to obtain the optimized orbitals required for accurately determining the isotropic HFCC. For the scalar-relativistic effects, we initially derived and implemented the Douglas-Kroll-Hess (DKH) hyperfine coupling operators up to the third order (DKH3) by using the direct transformation scheme. A set of 4d transition-metal radicals consisting of Ag atom, PdH, and RhH2 were chosen as test cases. Good agreement between the isotropic HFCC values obtained from DMRG/DKH3 and experiment was archived. Because there are no available gas-phase values for PdH and RhH2 radicals in the literature, the results from the present high-level theory may serve as benchmark data.

A simple and highly sensitive method for the determination of caffeine in energy beverages was developed and validated. Sample preparation utilizing solid-phase extraction (SPE) was simple and reliable. Separation by isocratic ultra-high performance liquid chromatography (UPLC) with a reversed-phase C18 column was performed within 6 min. The use of SPE with UPLC coupled with electrospray ionization-multiple tandem mass spectrometry detection (ESI-MSn) was accurate, reproducible, and validated for the determination of caffeine in energy drink matrices. The limit of quantification for caffeine was approximately 2.1 ng mL−1. The intra-day and inter-day precisions were less than 4%, and the accuracy of the measurements was between 85.1% and 93.2%. Results for caffeine concentrations in eighteen beverages were compared to the values on the labels. This paper describes the first use of the UPLC–ESI-ion trap MSn technique for quality-control purposes of caffeine present in energy drinks.

A fast and highly sensitive method for the quantification of ketoconazole in antidandruff emulsion formulas was developed and validated. Sample preparation utilizing solid-phase extraction was a simple and reliable method for extracting both ketoconazole and its internal standard miconazole from the samples: 97 ± 3% for ketoconazole and 93 ± 4% for the internal standard. The separation by isocratic ultra-high performance liquid chromatography and multiple-stage mass spectrometry with a reversed-phase C18 column was performed within 10 min. The method, which operated in a selective reaction monitoring mode specific to ketoconazole, was validated for quantitative use. The intra- and inter-day precision values were

Cyclosporin A (CyA), a cyclic undecapeptide produced by a number of fungi, contains 11 unusual amino acids, and has been one of the most commonly prescribed immunosuppressive drugs. To date, there are over sixty different analogs reported as congeners and analogs resulting from precursor-directed biosynthesis, human CYP-mediated metabolites, or microbial bio-transformed analogs. However, there is still a need for more structurally diverse CyA analogs in order to discover new biological potentials and/or improve the physicochemical properties of the existing cyclosporins. As a result of the complexity of the resulting mass spectrometric (MS) data caused by its unusual amino acid composition and its cyclic nature, structural characterization of these cyclic peptides based on fragmentation patterns using multiple tandem MS analyses is challenging task. Here, we describe, an efficient HPLC-ESI-ion trap MS(n) (up to MS(8)) was developed for the identification of CyA and CyC, a (Thr(2))CyA congener in which L-aminobutyric acid (Abu) is replaced by L-threonine (Thr). In addition, we examined the fragmentation patterns of a CyA analog obtained from the cultivation of a recombinant Streptomyces venezuelae strain fed with CyA, assigning this analog as (γ-hydroxy-MeLeu(6))CyA (otherwise, known as an human CYP metabolite AM6). This is the first report on both the MS(n)-aided identification of CyC and the structural characterization of a CyA analog by employing HPLC-ESI-ion trap MS(n) analysis.

A simple and rapid method for the determination of coenzyme Q10 in edible cosmetics was developed and validated. Cleanup utilizing a solid-phase extraction technique provided a simple and reliable method for extracting both coenzyme Q10 and its internal standard coenzyme Q9 from the lecithin-rich samples with efficiencies of 92 ± 4% and 89 ± 3%, respectively. The separation of analytes through a reversed-phase C18 column was carried out within 10 min. By employing authentic coenzyme Q10 and the internal standard spiked into blank matrices, the combined use of solid phase extraction cleanup and chromatographic separation coupled with electrospray ionization-tandem mass spectrometry detection was shown to be sufficiently accurate to detect coenzyme Q10 in edible cosmetic matrices. The limit of quantification of coenzyme Q10 determined by the validated method was 54 ng/g in the matrices. In addition, the intra- and inter-day precision was

We have used the non-equilibrium Green’s function in combination with the density functional theory to investigate the quantum transport properties of the molecular junctions including a terminated benzene ring directly coupled to surface of metal electrodes (physisorption). The other side of molecule was connected to electrode via thiolate bond (chemisorption). Two different electrodes have been studied, namely Cu and Al. Rectification and negative differential resistance behavior have been observed. We found that the electron transport mechanism is affected by the nature of benzene–electrode coupling. In other words, the transport mechanism depends on the nature of metallic electrode. Changing from sp - to sd -metallic electrode, the molecular junction changes from the Schottky to p–n junction-like diode. The transmission spectra, projected density of state, molecular projected self-consistent Hamiltonian, transmission eigenchannel, and Muliken population have been analyzed for explanation of electronic transport properties. Understanding the transport mechanism in junction having direct coupling of π -conjugate to electrode will be useful to design the future molecular devices.

RASSF enzymes act as key apoptosis activators and tumor suppressors, being downregulated in many human cancers, although their exact regulatory roles remain unknown. A key downstream event in the RASSF pathway is the regulation of MST kinases, which are main effectors of RASSF-induced apoptosis. The regulation of MST1/2 includes both homo- and heterodimerization, mediated by helical SARAH domains, though the underlying molecular interaction mechanism is unclear. Here, we study the interactions between RASSF1A, RASSF5, and MST2 SARAH domains by using both atomistic molecular simulation techniques and experiments. We construct and study models of MST2 homodimers and MST2-RASSF SARAH heterodimers, and we identify the factors that control their high molecular stability. In addition, we also analyze both computationally and experimentally the interactions of MST2 SARAH domains with a series of synthetic peptides particularly designed to bind to it, and hope that our approach can be used to address some of the challenging problems in designing new anti-cancer drugs., Author Summary We model the conformational changes and protein-protein interactions of enzymes involved in signaling along the Hippo pathway—a key molecular mechanism that controls the process of programmed cell death in eukaryotic cells, including cells affected by cancer. Combining modern computational modeling techniques with experimental information from X-ray crystallography and systems biology studies, can unveil detailed molecular interactions and lead to novel drugs. Here, we study the atomistic mechanisms and interactions between MST2 and RASSF-type kinases, through their respective SARAH domains—highly conserved, long, terminal α-helices, which play essential roles in the activation of MST kinases and, therefore, in modulating apoptosis. In spite of their key roles in mediating cell signaling pathways, there is little structural information available for the RASSF SARAH domains and their dimerization with the MST2 SARAH domains. In particular, the RASSF1A crystal structure is not available yet. Here, we model, refine and validate atomistic structural models of dimers of the RASSF1A and MST2 SARAH domains, studying the interaction and the dynamic behavior of these molecular complexes using homology modeling, docking and full atomistic molecular dynamics simulations. Experimentally, we validate our approach by designing a novel peptide that can disrupt effectively MST2 homo and hetero SARAH dimers.

Kinetics of a recombinant uridine diphosphate-glucose: sterol glycosyltransferase from Micromonospora rhodorangea ATCC 27932 (MrSGT) were studied using a number of sterols (including phytosterols) as glycosyl acceptors. The lowest K m value and the highest catalytical efficiency (k cat/K m) were found when β-sitosterol was the glycosyl acceptor in the enzymatic reaction. In contrast to the enzyme’s flexibility toward the glycosyl acceptor substrate, this recombinant enzyme was highly specific to uridine diphosphate (UDP)-glucose as the donor substrate. Besides, the UDP-glucose-dependent MrSGT was able to attach one glucose moiety specifically onto the C-3 hydroxyl group of other phytosterols such as fucosterol and gramisterol, yielding stereo-specific fucosterol-3-O-β-d-glucoside and gramisterol-3-O-β-d-glucoside, respectively. Based on kinetic data obtained from the enzyme’s reactions using five different sterol substrates, the significance of the alkene (or ethylidene) side chains on the C-24 position in the sterol scaffolds was described and the possible relationship between the substrate structure and enzyme activity was discussed. This is the first report on the enzymatic bioconversion of the above two phytosteryl 3-O-β-glucosides, as well as on the discovery of a stereospecific bacterial SGT which can attach a glucose moiety in β-conformation at the C-3 hydroxyl group of diverse sterols, thus highlighting the catalytic potential of this promiscuous glycosyltransferase to expand the structural diversity of steryl glucosides.

A uridine diphosphate-glucose:sterol glycosyltransferase-encoding gene was isolated and cloned from the established fosmid library of Micromonospora rhodorangea ATCC 27932 that usually produces the aminoglycoside antibiotic geneticin. The gene consists of 1,185 base pairs and encodes a 41.4 kDa protein, which was heterologously expressed in Escherichia coli BL21(DE3). In silico analyses of the deduced gene product suggested that it is a member of the family 1 glycosyltransferases. The recombinant protein MrSGT was able to catalyze the transfer of a glucosyl moiety onto the C-3 hydroxy function in sterols (β-sitosterol, campesterol, and cholesterol), resulting in the corresponding steryl glucosides (β-sitosterol-3-O-β-D-glucoside, campesterol-3-O-β-D-glucoside, and cholesterol-3-O-β-D-glucoside). This enzyme prefers phytosterols to cholesterol, and also shows substrate flexibility to some extent, in that it could recognize a number of acceptor substrates.

In this study, an efficient high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-ion trap-tandem mass spectrometry (MS/MS) was developed for the identification of the biosynthetic congeners involved in the aminocyclitol aminoglycosidic fortimicin pathway from Micromonospora olivasterospora fermentation. The usage of both acid extraction (pH ∼2.5) followed by an cationic-exchanging SPE cleanup and pentafluoropropionic acid mediated ion-pairing chromatography with ESI-ion trap-MS/MS detection was determined to be sufficiently practical to profile the fortimicin (FOR) congeners produced in a culture broth. The limit of the quantification for the fortimicin A (FOR-A) standard spiked in the culture broth was ∼1.6 ng mL(-1). The average recovery rate was 93.6%, and the intra- and inter-day precisions were

A newly reduced macrocyclic lactone antibiotic streptogramin A, 5,6-dihydrovirginiamycin M1 was created by feeding virginiamycin M1 into a culture of recombinant Streptomyces venezuelae. Its chemical structure was spectroscopically elucidated, and this streptogramin A analogue showed twofold higher antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA) compared with its parent molecule virginiamycin M1. Docking studies using the model of streptogramin A acetyltransferase (VatA) suggested that the newly generated analogue binds tighter with overall lower free energy compared with the parent molecule virginiamycin M1. This hypothesis was validated experimentally through the improvement of efficacy of the new analogue against MRSA strains. The biotransformation approach presented herein could have a broad application in the production of reduced macrocyclic molecules.

Natural rubber-degrading microorganisms were isolated from waste of rubber processing factory in Cam Thuy of Vietnam. Four of them belong to Streptomyces sp. that showed the higher abilities for natural rubber degradation than the others. They are able to use both deproteinised natural rubber (DPNR) and synthetic rubber cis-1,4-polyisoprene (SR) as a sole source of carbon. Gel permeation chromatography (GPC) analysis revealed that these strains degraded DPNR and SR to low-molecular-weight products. The growth of isolates occurs essentially on the latex glove pieces after one month of incubation in mineral salt medium. The total nitrogen contents of glove pieces were determined using Kjeldahl method, which were 10-20 times higher than that in un-inoculated sample. Moreover, the degradation was also confirmed by observing the growth of isolates on glove’s surface using scanning electron microscopy (SEM).

The medicinal mushroom Agaricus blazei produced high amounts of laccase (up to 5,000 units l(-1)) in a complex, agitated liquid medium based on tomato juice, while only traces of the enzyme (100 units l(-1)) were detected in synthetic glucose-based medium. Purification of the enzyme required three chromatographic steps, including anion and cation exchanging. A. blazei laccase was expressed as a single protein with a molecular mass of 66 kDa and an isoelectric point of 4.0. Spectroscopic analysis of the purified enzyme confirmed that it belongs to the "blue copper oxidases". The enzyme's pH optimum for 2,6-dimethoxyphenol (DMP) and syringaldazine was pH 5.5; but for 2,2'-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS) no distinct pH optimum was observed (highest activity at the lowest pH tested). Purified laccase was stable at 20 degrees C, pH 7.0 and pH 3.0, but rapidly lost its activity at 40 degrees C or pH 10. Sodium chloride strongly inhibited the enzyme activity, although the inhibition was completely reversible. The following kinetic constants were determined (K(m), k(cat)): 63 microM, 21 s(-1) for ABTS, 4 microM, 5 s(-1) for syringaldazine, 1,026 microM, 15 s(-1) for DMP and 4307 microM, 159 s(-1) for guaiacol. The results show that--in addition to the wood-colonizing white-rot fungi--the typical litter-decomposing basidiomycetes can also produce high titers of laccase in suitable liquid media.

The phosphoinositide 3-kinase (PI3K)-Akt network is tightly controlled by feedback mechanisms that regulate signal flow and ensure signal fidelity. A rapid overshoot in insulin-stimulated recruitment of Akt to the plasma membrane has previously been reported, which is indicative of negative feedback operating on acute timescales. Here, we show that Akt itself engages this negative feedback by phosphorylating insulin receptor substrate (IRS) 1 and 2 on a number of residues. Phosphorylation results in the depletion of plasma membrane-localised IRS1/2, reducing the pool available for interaction with the insulin receptor. Together these events limit plasma membrane-associated PI3K and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) synthesis. We identified two Akt-dependent phosphorylation sites in IRS2 at S306 (S303 in mouse) and S577 (S573 in mouse) that are key drivers of this negative feedback. These findings establish a novel mechanism by which the kinase Akt acutely controls PIP3 abundance, through post-translational modification of the IRS scaffold., eLife digest For the body to work properly, cells must constantly ‘talk’ to each other using signalling molecules. Receiving a chemical signal triggers a series of molecular events in a cell, a so-called ‘signal transduction pathway’ that connects a signal with a precise outcome. Disturbing cell signalling can trigger disease, and strict control mechanisms are therefore in place to ensure that communication does not break down or become erratic. For instance, just as a thermostat turns off the heater once the right temperature is reached, negative feedback mechanisms in cells switch off signal transduction pathways when the desired outcome has been achieved. The hormone insulin is a signal for growth that increases in the body following a meal to promote the storage of excess blood glucose (sugar) in muscle and fat cells. The hormone binds to insulin receptors at the cell surface and switches on a signal transduction pathway that makes the cell take up glucose from the bloodstream. If the signal is not engaged diseases such as diabetes develop. Conversely, if the signal cannot be adequately switched of cancer can develop. Determining exactly how insulin works would help to understand these diseases better and to develop new treatments. Kearney et al. therefore set out to examine the biochemical ‘fail-safes’ that control insulin signalling. Experiments using computer simulations of the insulin signalling pathway revealed a potential new mechanism for negative feedback, which centred on a molecule known as Akt. The models predicted that if the negative feedback were removed, then Akt would become hyperactive and accumulate at the cell’s surface after stimulation with insulin. Further manipulation of the ‘virtual’ insulin signalling pathway and studies of live cells in culture confirmed that this was indeed the case. The cell biology experiments also showed how Akt, once at the cell surface, was able to engage the negative feedback and shut down further insulin signalling. Akt did this by inactivating a protein required to pass the signal from the insulin receptor to the rest of the cell. Overall, this work helps to understand cell communication by revealing a previously unknown, and critical component of the insulin signalling pathway.